JP2008037872A - N-terminally monopegylated human growth hormone conjugate, process for their preparation, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chemically modified derivative of human growth hormone (hGH) supposed to have hGH activity prolonging for a much longer period compared with unmodified hGH and enabling reduced dose and therapeutic frequency in therapeutic scheme. <P>SOLUTION: The invention provides a chemically modified protein prepared by attaching a butyraldehyde moiety of a polyethylene glycol to the N-terminal phenylalanine of human growth hormone (hGH), and a method of its use for the treatment and/or prevention of diseases or disorders in which use of growth hormone is beneficial. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

This application claims priority to US patent application Ser. No. 10 / 77,895 filed Feb. 4, 2004, which is incorporated by reference in its entirety as if set forth herein. To do.

TECHNICAL FIELD OF THE INVENTION
[001] The present invention relates to chemical modifications involving PEGylation of hGH and agonist variants thereof, thereby altering the chemical and / or physiological properties of human growth hormone (hGH). The PEGylated hGH can have increased plasma residence time, decreased clearance rate, improved stability, decreased antigenicity, decreased PEGylation heterogeneity, or combinations thereof. The invention also relates to a method for the modification of hGH. In addition, the present invention relates to pharmaceutical compositions containing modified hGH. A further aspect is the use of modified hGH for the treatment of growth and developmental disorders.

Background of the Invention
[002] Human growth hormone (hGH) is a protein comprising a single chain of 191 amino acids cross-linked by two disulfide bridges, the monomer form having a molecular weight of 22 kDa. Human GH is secreted by the pituitary gland, which can also be produced by recombinant genetic engineering. hGH causes growth in all body tissues that can grow. hGH not only promotes human growth but also plays an important role in maintaining normal body composition, anabolism, and lipid metabolism (K. Barneis. And U. Keller, Baillieres Clin Endocrinlo. Metab. 10: 337 (1996)).

[003] Recombinant hGH has become commercially available for several years. Two types of therapeutically useful recombinant hGH preparations are on the market: natural types such as Genotropin® or Neuropin®, and analogs with an additional methionine residue at the N-terminus For example, Somatonorm (registered trademark) can be mentioned. hGH is used to stimulate height loss (axial growth) in patients with hypopituitarism dwarfism, also known as growth hormone deficiency (GHD), or Turner syndrome, but during gestation In contrast, for long-term treatment of growth failure in children born short (SGA), for Prader-Willi syndrome (PWS), chronic renal failure (CRI), exhaustion with AIDS, and treatment of patients with aging, otherwise The application of was also suggested. Patients with adult growth hormone deficiency (aGHD) include various problems such as increased fat mass, decreased lean body mass and extracellular fluid, and decreased bone density, dyslipidemia, and cardiovascular dysfunction , Having a characteristic change in body composition. Many of these problems are ameliorated by hGH replacement therapy (J. Verhelst J and R. Abs. Drugs .; 62: 2399 (2002)).

[004] The main biological effects of growth hormone (GH) are the promotion of growth in young mammals and the maintenance of older mammalian tissues. Affected organ systems include skeleton, connective tissue, muscle, and internal organs such as liver, intestine, and kidney. Growth hormone exerts their effects through interaction with specific receptors on the target cell membrane. hGH is a member of a family of homologous hormones including placental lactogen, prolactin, and other gene and species variants of growth hormone (Nicoll, CS, et al. (1986) Endocrine Reviews 7: 169). hGH, among these, exhibits specificity for a wide range of species and has been cloned somatogenic receptor (Leung, DW, et al
[1987] Nature 330; 537), or prolactin receptor (Boutin, JM, et al. [1988]
Cell; 53: 69) is different in that it binds to one of The cloned gene for hGH is expressed in secreted form in E. coli (Chang, CN, et al. [1987] Gene 55: 189), and its DNA and amino acid sequences have been reported (Goeddel, et al. [1979 ) Nature 281: 544; Gray, et al. [1985] Gene 39: 247).

[005] Human growth hormone (hGH) is involved in many of the regulation of normal human growth and development. This pituitary hormone exhibits numerous biological effects, including height gain (somatogenesis), lactation, macrophage activation, insulin-like effects and especially diabetes-inducing effects (Chawla, R , K. (1983) Ann. Rev. Med. 34, 519; Edwards, CK et al. (1988) Science 239, 769; Thomer, M. 0., et al. (1988) J. Clin. Invest. 81 : 745). Growth hormone deficiency in children results in dwarfism, which has been successfully treated for over 10 years by the administration of hGH from outside the body.

[006] Similar to children in adults, hGH maintains normal body composition through increased nitrogen retention and stimulation of skeletal muscle growth, and through the mobilization of body fat. Visceral adipose tissue is particularly responsive to hGH. In addition to increasing lipolysis, hGH reduces triglyceride incorporation into body fat stores. Serum concentrations of IGF-I (insulin-like growth factor-I) and IGFBP3 (insulin-like growth factor binding protein 3) are increased by hGH.

[007] Human growth hormone (hGH) is a single-chain polypeptide consisting of 191 amino acids (molecular weight 21,500). Disulfide bonds bridge positions 53 and 165, and positions 183 and 189. Niall, Nature, New Biology, 230: 90 (1971).
hGH is a potent anabolic agent, especially by retention of nitrogen, phosphorus, potassium and calcium. Treatment of hypophysectomized rats with GH can restore at least a portion of the growth rate of the rats. Moore et al., Endocrinology 122: 2920-2926 (1988). Among them, the most prominent effect in subjects with hypopituitarism (GH deficiency) is accelerated axial growth of epiphyseal cartilage, resulting in increased height. Kaplan, Growth Disorders
in Children and Adolescents (Springfield, IL: Charles C. Thomas, 1964).

[008] hGH causes a variety of physiological and metabolic effects in a variety of animal models, including axial bone growth, lactation, macrophage activation, insulin-like and diabetogenic effects, and the like. (RK Chawla et al., Annu. Rev. Med. 34: 519 (1983);
0. GP Isaksson et al., Annu. Rev. Physiol. 47, 483 (1985); CK Edwards et al., Science 239, 769 (1988); M. 0. Thomer and ML Vance, J. Clin. Invest. 82: 745 (1988); JP Hughes and HG Friesen, Ann. Rev. Physiol. 47: 469 (1985)).
GH secretion was reported to decline with age, particularly in postmenopausal women. Millard et al., Neurobiol. Aging, 11: 229-235 (1990); Takahashi et al., Neuroendocrinology M, L6- 137-142 (1987). See also Rudman et al., J. Clin. Invest., 67: 1361-1369 (1981) and Blackman, Endocrinology and Aging, 16: 981 (1987). Furthermore, there are reports that some of the manifestations of aging, including reduced slow fat body weight, increased adipose tissue mass, and thinning of the skin, can be reduced by GH treatment three times a week. See, for example, Rudman et al., N. Eng. J. Med., 323: 1-6 (1990), and the accompanying paper by Dr. Vance (pp. 52-54) in the same journal publication. These biological effects are h
Derived from the interaction between GH and specific cellular receptors. Two different human receptors, the hGH liver receptor (DW Leung et al., Nature 330: 537 (1987)), and the human prolactin receptor (JM Boutin et al., Mol. Endocrinology. 3: 1455 (1989) )) Was cloned. However, there appear to be others including the human placental lactogen receptor (M. Freemark, M. Comer, G. Komer, and S. Handwerger, Endocrinol. 120: 1865 (1987)). These homologous receptors contain a sugar-linked extracellular hormone-binding domain, a single transmembrane domain, and a cytoplasmic domain that vary considerably in sequence and size. One or more receptors are thought to play a critical role in the physiological response to hGH.

[009] It is generally observed that physiologically active proteins administered into the body can only show their pharmacological activity for a short period of time due to their fast clearance rate in the body. Yes. Furthermore, the relative hydrophobicity of these proteins appears to limit their stability and / or solubility.

[0010] Several methods have been proposed to chemically modify proteins with water-soluble polymers for the purpose of reducing the clearance rate of therapeutic proteins, improving stability, or eliminating antigenicity. It was done. This type of chemical modification appears to effectively block proteolytic enzymes from physical contact with the protein backbone itself, thereby preventing denaturation. Chemical conjugation of certain water-soluble polymers appears to effectively reduce renal clearance due to the increased hydrodynamic volume of the molecule. Additional benefits include increased stability and circulation time of therapeutic proteins, increased solubility, and decreased immunogenicity under certain circumstances. Poly (alkene oxide), especially poly (ethylene glycol) (PEG), is one such chemical moiety that has been used in the preparation of therapeutic protein products (the verb “pegylated” is Means to attach at least one PEG molecule). The binding of poly (ethylene glycol) has been shown to prevent proteolysis, and Sada, et al., J. Fermentation Bioengineering 71: 137-139
(1991), a method for attaching certain poly (ethylene glycol) moieties can be utilized. U.S. Pat. No. 4,179,337 issued Dec. 18, 1979, Davis et al., "Non-Immunogenic Polypeptides,"; and U.S. Pat. No. 4,002 issued Jan. 11, 1977. , 531, Royer, "Modifying Enzymes with Polyethylene
See Glycol and Product Produced representative, "for a summary see Abuchowski et al., Enzymes as Drugs (JS Holcerberg and J. Roberts, eds. Pp. 367-383 (1981)).

[0011] Other water-soluble polymers have also been used, such as ethylene glycol / polyethylene glycol copolymers, carboxymethyl cellulose, dextran, poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (-1,3-dioxolane), Poly (-1
, 3,6-trioxane), ethylene / maleic anhydride copolymers, poly-amino acids (either homopolymers or random copolymers).

[0012] Numerous examples of pegylated therapeutic proteins have been described. ADAMGEN®, a pegylated formulation of adenosine deaminase, is approved for the treatment of severe combined immunodeficiency diseases. ONCASPAR®, or pegylated L-asparaginase, is approved for the treatment of all patients with hypersensitivity. PEGylated superoxide dismutase has been clinically attempted for the treatment of head trauma. Pegylated α-interferon (US Pat. Nos. 5,738,846, 5,382,657) has been approved for the treatment of hepatitis; pegylated glucocerebrosidase and pegylated hemoglobin have been preclinically tested Has been reported. Another example is poly (added to IL-6
PEGylated IL-6 of EF 0 442 724 entitled “Modified hIL-6” which discloses an (ethylene glycol) molecule.

[0013] Another specific therapeutic protein that has been chemically modified is granulocyte colony stimulating factor (G-CSF). G-CSF induces rapid proliferation and release of neutrophilic granulocytes into the bloodstream, thereby providing a therapeutic effect in combating infection. European Patent Application EP 0 401 384 entitled “Chemically Modified Granulocyte Colony Stimulating Factor” published on December 12, 1990, is a material and method for preparing G-CSF that binds poly (ethylene glycol) molecules. Is described. Modified G-CSF and its analogs also describe the use of various G-CSF and derivatives conjugated by covalent bonding with water-soluble particle polymers such as poly (ethylene glycol), “Continuous Release Pharmaceutical Compositions Comprising
EP 0 473 268, published March 4, 1992, entitled “A Polypeptide Covalently Conjugated To A Water Soluble Polymer”. A modified polypeptide having human granulocyte colony-stimulating factor activity is EP 0 335 423 published on Oct. 4, 2010. Provided in US Patent 5,824,784 is a method for modifying a protein or analog thereof at the N-terminus, and a novel The resulting composition comprising a chemically modified G-CSF composition at the N-terminus of US Patent No. 5,824,778 discloses chemically modified G-CSF.

  [0014] With respect to poly (ethylene glycol), various means have been used to attach poly (ethylene glycol) molecules to proteins. In general, poly (ethylene glycol) molecules are connected to the protein via reactive groups found on the protein.

[0015] An amino group, such as an amino group on a lysine residue or at the N-terminus, is convenient for such attachment. For example, Royer (US Pat. No. 4,002,531, described above) states that reductive alkylation was used to attach a poly (ethylene glycol) molecule to an enzyme. Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) report the modification of CD4 immunoadhesin with monomethoxypoly (ethylene glycol) aldehyde via reductive alkylation. The authors report that 50% of CD4-Ig was MePEG-modified under conditions where the degree of PEGylation could be controlled. 137 pages of the same document. The authors also described in vitro the modified CD4-Ig (against protein gp 120) in vitro.
Has been reported to decrease at a rate correlated to the degree of MePEGylation. 199
U.S. Pat. No. 4,904,584, issued Feb. 27, 0 0, Shaw describes a certain number of lysine residues in proteins relating to the attachment of poly (ethylene glycol) molecules via reactive amino groups. It relates to group modification.

[0016] WO 93/00109 is for stimulating mammalian or avian GH-responsive tissues, including maintaining a continuous effective plasma GH concentration for 3 days or more. Regarding the method. One way to achieve such plasma concentrations is stated to be by the use of GH coupled with a macromolecular substance such as PEG (polyethylene glycol). Coupling of macromolecules with substances is stated to provide improved half-life. Pegylated human growth hormone is described in WO 93/00109
Reported using mPEG aldehyde-5000 and mPEG N-hydroxysuccinimidyl ester (mPEG-NHS-5000). The use of mPEG-NHS resulted in a heterogeneous mixture of multiple PEGylated forms of hGH. WO 93/0
0109 also discloses the use of mPEG-maleimide to pegylate cysteine hGH variants.

[0017] WO 99/03887 discloses PEGylated cysteine mutant growth hormone. This conjugate, called BT-005, is more effective in stimulating weight gain in rats with growth incompetence, and is intended to have a longer half-life than hGH.

[0018] Pegylated human growth hormone was also reported in Clark et al. (Journal of Biological Chemistry 271: 21969-21977, 1996) using a succinimidyl ester of carboxymethylated PEG. Clark et al. Describe increased size derivatives of hGH using mPEG-NHS-5000 that is selectively conjugated to primary amines. Increasing the level of PEG modification decreased its affinity for the receptor and increased the EC ₅₀ in cell-based assays up to 1500-fold. Olson et al., Polymer Preprints 38: 568-569, 1997, N-hydroxysuccinimide (NHS)
The use of PEG and succinimidyl propionate (SPA) PEG is disclosed to result in multiple PEGylated hGH species.

[0019] WO 94/20069 describes pegylated hGH as part of a formulation for pulmonary delivery.
Is disclosed.
[0020] US Patent 4,179,337 discloses a method for pegylating enzymes and hormones to obtain physiologically active non-immunogenic water-soluble polypeptide conjugates. GH is mentioned as an example of a hormone that PEGylates.

  [0021] EP 458064 A2 discloses pegylation of cysteine residues introduced or naturally occurring in somatotropin. EP 458064 A2 further describes the incorporation of two cysteine residues in a loop called the omega loop which is said to be located at residues 102-112 of wild type bovine somatotropin. More specifically, EP 458064 A2 discloses substitution of residues 102 to 112 of bovine somatotropin from Ser to Cys and Tyr to Cys, respectively.

[0022] WO 95/11987 proposes the attachment of PEG to the thio group of any cysteine residue present in the parent molecule or introduced by site-directed mutagenesis. WO
95/11987 relates to pegylation of the protease nexin-1, but also proposes general pegylation of hGH and other proteins.

[0023] WO 99/03887 discloses, for example, growth hormone modified by insertion of an additional cys at the serine residue 25 and conjugating PEG to the introduced cysteine residue.

[0024] WO 00/42175 relates to a method for making a protein containing a free cysteine residue for attachment of PEG. WO 00/42175 discloses the following muteins of hGH: T3C, S144C and T148C, and their cysteine pegylation.

[0025] WO 97/11178 (as well as US 5849535, US 6000049)
1 and US 6022711) relate to the use of GH variants as agonists or antagonists of hGH. WO 97/11178 also discloses PEGylation of lysine and hGH including lysine introduction or substitution (eg K168A and K172R). WO 97/11178 also discloses the substitution G120K.

[0026] WO 03/044056 discloses various pegylated hGH species, including branched 40K PEG aldehyde hGH conjugates.
[0027] Previous reports of PEGylated hGH require multiple PEG conjugations, which will result in undesirable heterogeneous products, but the described renal filtration cut-off values This is to obtain a hydrodynamic volume greater than 70K molecular weight (Knauf, MJ et al, J. Biol. Chem. 263: 15064-15070, 1988).

[0028] Current administration of rhGH is performed daily for an extended period of time, and therefore less frequent administration is highly desirable. HGH molecules with longer circulatory half-lives will reduce the number of doses required and may provide more optimal therapeutic hGH levels with concomitant improved therapeutic effects.

[0029] Despite several attempts to PEGylate hGH, there remains an unmet need for PEGylated hGH molecules with suitable properties to be viable commercial products. Yes. The present invention provides PEG-hGH conjugates having a single PEG that is predominantly attached to the N-terminal phenylalanine of hGH, providing advantages over other PEG-hGH conjugates. Using mPEG aldehyde-5000 or mPEG N-hydroxysuccinimidyl ester (mPEG-NHS-5000), multiple low molecular weights (5 Kd) at the α- or ε-amino site (N-terminal and 9 lysines in hGH). ) Of WO 93/00109, Clark et al. (Journal
of Biological Chemistry 271: 21969-21977, 1996, and Olson et al. (Polymer Preprints 38: 568-569, 1997). This method results in a heterogeneous population. For example, hGH with 9 lysines has some molecules with 10 attached PEGs, some molecules with 9 molecules, some molecules with 8 molecules, some molecules with 7 molecules, Some molecules with six, some molecules with five, some molecules with four, some molecules with three, some molecules with two, one molecule with one It seems to have a part molecule, some molecules with zero. And among molecules with several PEGs, it appears that PEGs are not attached to the same position in different molecules. This resulting heterogeneity has the disadvantage that it is difficult, expensive and very reproducible to make, purify and characterize the conjugates during the development of therapeutic products. Another approach (WO 00/42
175) used an hGH variant containing a free cysteine residue for conjugation of PEG. However, this approach can result in a protein that is not correctly folded, with the wrong pair of disulfide bonds, and a heterogeneous PEGylated product with PEG attached to some or all of the cysteines. . Having multiple PEGs attached to multiple sites would result in molecules with more labile bonds between PEG and the various sites, which could become dissociated at different rates. is there. This makes it difficult to accurately predict the pharmacokinetics of the product and results in inaccurate administration. Heterogeneous products can also have undesirable problems in obtaining regulatory approvals for therapeutic products.

  [0030] Therefore, it would be desirable to have a pegylated hGH molecule with a single PEG attached to a single site. The present invention addresses this need in several ways.

Summary of the Invention
[0031] The present invention relates to chemically modified hGH and agonist variants thereof, which include decreased clearance rate, increased plasma residence time, increased stability, improved solubility, and decreased antigenicity. It has at least one improved chemical or physiological property that is more selected but not limited. Thus, as described in more detail below, the present invention involves chemically modifying polypeptides, including but not limited to hGH and agonist variants thereof, and using the butyraldehyde portion of poly (ethylene glycol). It has several aspects regarding all specific modifications.

  [0032] The present invention also relates to a method of generating chemically modified hGH and agonist variants thereof. In particular, the present invention relates to a method for producing chemically modified hGH using butyraldehyde, which results in a higher selectivity than binding to the N-terminus.

[0033] The present invention also relates to compositions that include chemically modified hGH and agonist variants thereof, alone or in combination with another therapeutic agent.
[0034] The present invention also provides for the prevention of disorders and / or diseases for which GH treatment is useful, using the chemically modified hGH and agonist variants thereof of the present invention alone or in combination with another therapeutic agent. And / or for use in therapy.

Detailed description
[0041] hGH and agonist variants thereof are described in US Pat. No. 4,658,021 (methionyl human growth hormone-Met-1-191 hGH) and US Pat.
2 is a member of the family of recombinant proteins described in 2. These recombinant products and methods of use are described in US Pat. Nos. 4,342,832, 4,601,980;
4,898,830; US Pat. No. 5,424,199; and US Pat. No. 5,795,745.

[0042] Any purified, isolated hGH or agonist variant thereof produced by host cells, eg, E. coli and animal cells transformed or transfected by using recombinant gene technology, is used in the present invention. It's okay. Additional hGH variants are described in US Pat. No. 6,143,523 and WO 92/09690 published on January 11, 1992. Among them, hGH produced by transformed E. coli or an agonist variant thereof is particularly preferred. Such hGH or agonist variants thereof may be obtained in large quantities with high purity and homogeneity. For example, hGH or agonist variants thereof are described in US Pat. No. 4,342,832, 4,601,980; US Pat. No. 4,898,830; US Pat. No. 5,424,199; and US Pat. No. 5,795,745. It may be prepared according to the disclosed method. The term “substantially having the following amino acid sequence” means that the amino acid sequence described above is one or more of the above unless it causes any adverse dissimilarity in the function of hGH or an agonist variant thereof. It means that an amino acid change (deletion, addition, insertion or substitution) may be included. HGH or an agonist mutation thereof substantially having an amino acid sequence comprising at least one lysine, aspartic acid, glutamic acid, unpaired cysteine residue, free N-terminal α-amino group, or free C-terminal carboxyl group More preferably, the body is used.

[0043] The term " hGH polypeptide or hGH protein " as used herein is characterized by promoting growth in the growth phase and in maintaining normal body composition, anabolism and lipid metabolism. All hGH polypeptides, preferably from mammalian species, more preferably from human and murine species, as well as variants, analogs, orthologs, homologues and derivatives thereof, and Of fragments. Preferably, the term “hGH polypeptide or hGH protein” refers to the hGH polypeptide of SEQ ID NO: 1 and essentially the same biological activity (in the growth phase and maintaining normal body composition, anabolism and lipid metabolism) And its variants, homologues and derivatives that essentially exhibit growth). More preferably, the term “hGH polypeptide or hGH protein” refers to the polypeptide of SEQ ID NO: 1.

[0044] The term "hGH polypeptide variant" as used herein refers to a polypeptide that is derived from the same species but is different from a reference hGH polypeptide. In general, the amino acid sequences of the reference and variant are almost similar overall and the differences are limited so that they are identical in many regions. Preferably, the hGH polypeptide is at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference hGH polypeptide, preferably the hGH polypeptide of SEQ ID NO: 1. . With a polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence, the subject polypeptide sequence may comprise up to 5 amino acid changes for every 100 amino acids of the query amino acid sequence. Otherwise, the amino acid sequence of the subject polypeptide is intended to be identical to the query sequence. These changes from the reference sequence can be made at the amino- or carboxyl-terminal position of the reference amino acid sequence, or anywhere between the positions of both ends, ie individually between the residues in the reference sequence, or the reference sequence It may occur interspersed with any of one or more of the consecutive groups. The query sequence can be the entire amino acid sequence of the reference sequence, or any fragment identified as described above.

  [0045] Such hGH polypeptide variants are naturally occurring variants, such as naturally occurring alleles encoded by one of several different forms of hGH that occupy a given locus on the chromosome of an organ. It may be an isoform encoded by a variant of the gene, or a naturally occurring splice variant originating from one initial transcript. Alternatively, hGH polypeptide variants may be variants that are not known to occur naturally and can be made using mutation techniques known in the art.

  [0046] It is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus of a bioactive peptide or protein without substantially losing biological function. (See, for example, Ron et al., (1993), Biol Chem., 268 2984-2988; the disclosure of which is hereby incorporated by reference in its entirety).

[0047] It will also be appreciated by those skilled in the art that the amino acid sequence of some of the hGH polypeptides can be altered without significant effect on the structure or function of the protein. Such mutations include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity. For example, a guide on how to perform phenotypically silent amino acid substitutions is available in Bowie et al.
(1990), Science 247: 1306-1310, which is hereby incorporated by reference in its entirety, in which the authors study the resistance of amino acid sequences to changes. It shows that there are two major approaches.

[0048] The first method relies on an evolutionary process in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions with cloned hGH, and selection or screening to identify sequences that maintain functionality. These studies revealed that proteins are surprisingly resistant to amino acid substitutions. The authors also showed which amino acid changes are likely to be accepted at certain positions in the protein. For example, (structurally) the most buried amino acid residues require non-polar side chains, while only a few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al. (1990), supra, and references cited therein.

[0049] Typically recognized as conservative substitutions are substitutions of residues between the aliphatic amino acids Ala, Val, Leu and Phe to another residue; the hydroxyl residues Ser and Thr exchange, Asp and Glu exchange as acidic residues, substitution between amide residues Asn and Gln, exchange between basic residues Lys and Arg, and aromatic residues Phe, Tyr It is a replacement between. In addition, the following groups of amino acids generally show equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln
, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Ty
r, Trp, and His.

[0050] The term hGH polypeptide also includes analogs of hGH, orthologs, and / or
Or comprehensively includes all hGH polypeptides encoded by species homologues. As used herein, the term “hGH analog” performs the same function in each organism, but is not based on the structure of the origin that the origin of the organism had in common, and An unrelated organism, hGH. Rather, hGH analogs were generated separately and then evolved to perform the same function (or similar function). In other words, hGH analog polypeptides have completely different amino acid sequences but promote growth in the same biological activity, i.e. in the growth phase, and in maintaining normal body composition, anabolism and lipid metabolism. The polypeptide to be performed. As used herein, the term “hGH ortholog” refers to two sequences whose sequences are related to each other through a common homologous hGH in a species of origin, but have evolved to differ from each other. Different species of hGH. As used herein, the term “hGH homologue” refers to the hGH of different organisms that perform the same function in each organism and are based on a structure of origin that the origin of the organism had in common. Say. In other words, hGH homolog polypeptides have very similar amino acid sequences that promote growth in the same biological activity, i.e. in growth phase and in maintaining normal body composition, anabolism and lipid metabolism. It is a polypeptide. Preferably, the hGH polypeptide homologue is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to a reference hGH polypeptide, preferably the hGH polypeptide of SEQ ID NO: 1. Polypeptides exhibiting 96%, 97%, 98% or 99% identity may be defined.

  [0051] Thus, the hGH polypeptide according to the present invention may be: For example: (i) one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acids Residues may or may not be encoded by the genetic code: or (ii) one or more amino acid residues contain a substituent: or (iii) apart from hGH polypeptide Or a compound that increases the half-life of the polypeptide (eg, polyethylene glycol): or (iv) an additional amino acid, such as a peptide in the Ig Fc fusion region, or a leader or secretory sequence, or A sequence used for purification of the polypeptide or proprotein sequence into a topological form, Which is fused to a form of the above procedure, and the like.

[0052] hGH polypeptides may be monomeric or multimeric. The multimer may be a dimer, trimer, tetramer, or multimer comprising at least 5 monomeric polypeptide units. Multimers may also be homodimers or heterodimers. The multimers of the present invention may be obtained by hydrophobic, hydrophilic, ionic and / or covalent association (or binding) and / or indirectly linked, for example by liposome formation. May be. In one example, a covalent bond is present between heterogeneous sequences contained in a fusion protein containing an hGH polypeptide or fragment thereof (see, eg, US Pat. No. 5,478,925, the disclosure of which is hereby incorporated by reference) Which is incorporated by reference in its entirety by the specification.) In another example, an hGH polypeptide or fragment thereof is one or more poly-peptides that may be either an hGH polypeptide or a heterogeneous polypeptide. The peptide is linked through a peptide linker, such as those described in US Pat. No. 5,073,627 (incorporated herein by reference). Another method for preparing multimeric hGH polypeptides is known to promote multimerization of proteins obtained using techniques known to those skilled in the art, including those of WO 94/10308. Use of hGH polypeptides fused to leucine zipper or isoleucine zipper polypeptide sequences. In another example, the hGH polypeptide may be associated by interaction with a Flag® polypeptide sequence contained in a fusion hGH polypeptide containing a Flag® polypeptide sequence. . hGH multimers can also be synthesized by chemical techniques known in the art, such as linker molecules and linker molecule length optimization techniques known in the art (see, eg, US Pat. No. 5,478,925). Known in the art to form one or more intermolecular bridges between cysteine residues located within the sequence of the polypeptide desired to be contained in the multimer (eg, US patents). 5,478,925), addition of cysteine or biotin to the C-terminus or N-terminus of the hGH polypeptide, and to produce multimers containing one or more of these modified polypeptides. (See, for example, US Pat. No. 5,478,925), or 30 liposomes containing hGH multimers. Either operative (see for example U.S. Pat. No. 5,478,925), may be prepared using. The above disclosure is incorporated by reference in their entirety.

  [0053] As used herein, the term "hGH polypeptide fragment" refers to any peptide or polypeptide that includes a contiguous section of the amino acid sequence of an hGH polypeptide, preferably the polypeptide of SEQ ID NO: 1. Refers to a peptide.

[0054] More specifically, hGH polypeptide fragments are at least 6, at least 8 to 10, more preferably 12, 15, 20, 25, 30, 35, 40, 50, 60 of hGH polypeptides according to the present invention. 75, 100, 125, 150, 175, 191 consecutive amino acids. hGH polypeptide fragments may additionally be described as sub-genuses of hGH polypeptides comprising at least 6 amino acids, where “at least 6” is 6, and the polypeptide of SEQ ID NO: 1 Is defined as any integer between and the integer representing the C-terminal amino acid of the hGH polypeptide. Also included are species of hGH polypeptide fragments of at least 6 amino acids in length as described above, further specified in terms of their N-terminal and C-terminal positions. Included by the term “hGH polypeptide fragment” as an individual species are all hGH of at least 6 amino acids in length, as described above, which may also be specifically identified by N-terminal and C-terminal positions. It is a polypeptide fragment. That is, each combination of N-terminal and C-terminal positions that can be occupied by a fragment of at least six consecutive amino acid residue lengths on the sequence list or any given amino acid sequence of the present invention. include.

[0055] Alternatively, the above species of polypeptide fragments of the invention may be represented by the formula "a to b"
In particular, where "a" is equal to the amino acid position from the N-terminal end of the polynucleotide, and "b" is equal to the amino acid position from the C-terminal end; Further, where “a” is equal to an integer between 1 and the number of amino acids in the hGH polynucleotide sequence minus 6 and “b” is an integer between 7 and the number of amino acids in the hGH polynucleotide sequence Where “a” is an integer that is at least 6 less than “b”.

  [0056] The above hGH polypeptide fragments can be readily recognized using the above description and are therefore not listed individually for the purpose of not unnecessarily lengthening the specification. Furthermore, the above-mentioned fragments do not necessarily have hGH biological activity, but polypeptides having these activities are useful, for example, in immunoassays, in epitope mapping, epitope tagging, as vaccines, and as molecular weight markers. Thus, peptides having such activity are a preferred embodiment of the present invention. The above fragments may also be used to generate antibodies against specific portions of the polypeptide.

[0057] Also encompassed generically by the term "hGH polypeptide fragment" is a domain of hGH polypeptide. Such domains include leucine zippers, helix-turn-helix motifs, post-translational modification sites such as glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signals that direct secretion of the encoded protein. Linear or structural including, but not limited to, signal sequences encoding peptides, sequences involved in the regulation of transcription, such as homeoboxes, acidic stretches, enzyme active sites, substrate binding sites, and enzymatic cleavage sites Motifs and signatures may consequently be included. Such domains include specific biological activities such as DNA or RNA binding, protein secretion, transcriptional regulation, enzyme activity, substrate binding activity, etc. . . May be expressed.

  [0058] One domain generally includes between 3 and 191 amino acids. In a more preferred embodiment, the domain includes a number of amino acids that is any integer between 6 and 191. Domains may be synthesized using any method known to those of skill in the art, including those disclosed herein for the preparation of hGH polypeptides to generate anti-hGH antibodies. Methods for determining the amino acids that make up a domain with a particular biological activity include mutagenesis tests and assays for determining the biological activity to be tested.

  [0059] Particularly preferred fragments in the context of the present invention retain substantial biological activity, i.e. promote growth in the growth phase and in maintaining normal body composition, anabolism and lipid metabolism. HGH polypeptide.

[0060] Alternatively, polypeptides of the invention may be scanned for motifs, domains, and / or signatures in a database using any computer method known to those of skill in the art. A searchable database is Prosite (Hofmann et al., (1999) Nucl. Acids Res. 27: 215-219; Bucher and Bairoch (1994) Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman et al, Eds. , pp53-61, AAAIPress, Menlo Park), Pfam (Sonnhammer et al., (1997) Proteins 28 (3): 405-20; Henikoff et al., (2000) Nucleic Acids Res. 28 (1): 228-30 Bateman et al., (2000) Nucleic Acids Res. 28 (1): 263-6), Blocks (Henikoff et al., (2000) Electrophoresis 21 (9): 1700-6), Print (Attwood et al. , (1996) Nucleic Acids Res. 24 (1): 182-8), Prodom (Sonnhammer and Kahn (1994) Protein Sci. 3 (3): 482-92; Corpet et al.
(2000) Nucleic Acids Res. 28 (1): 267-9), Sbase (Pongor et al. (1993) Protein Eng.
6 (4): 391-5; Murvai et al., (2000) Nucleic Acids Res. 28 (1): 260-2), Smart (Schultz et al., (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864), Dali / FSSP (Holm and Sander (1996) Nucleic Acids Res. 24 (1): 206-9; Holm and Sander (1997) Nucleic
Acids Res. 25 (1): 231-4; Holm and Sander (1999) Nucleic Acids Res. 27 (1): 244-7), HSSP (Sander and Schneider (1991) Proteins 9 (1): 56-68. ), CATH (Orengo et al., (1997) Structure5 (8): 1093-108; Pearl et al., (2000) Biochem. Soc. Trans. 28 (2): 269-75), SCOP (Murzin et al , (1995) J. Mol. Biol. 247 (4): 536-40; Lo Conte et al., (2000) Nucleic Acids Res. 28 (1): 257-9), COG (Tatusov et al., (1997) Science 278, 631: 637; Tatusov et al., (2000) Nucleic Acids Res. 28 (1): 33-6), specific family databases and derivatives thereof (Nevill-Manning et al., (1998) Proc Natl. Acad. Sci. US A. 95, 5865-5871; Yona et al., (1999) Proteins 37 (3): 360-78; Attwood et al., (2000) Nucleic Acids Res. 28 (1) : 225-7), each of which is hereby incorporated by reference in its entirety. For a summary of available databases, see Nucleic Acid Research (2000) Vol. 28, No. 1, the disclosure of which is hereby incorporated by reference in its entirety.

[0061] The term "hGH polypeptide fragment" also includes inclusive fragments that carry the epitope. These epitopes can be antigenic epitopes or both antigenic and immunogenic epitopes. An immunogenic epitope is defined as the part of a protein that produces an antibody response in vivo when the polypeptide is an immunogen. On the other hand, the region of the polypeptide to which the antibody binds is defined as the antigen epitope. An epitope can include as few as 3 amino acids in a spatial conformation, which is unique to the epitope. In general, an epitope consists of at least 6 amino acids, and more often at least 8-10 such amino acids.

  [0062] A fragment carrying an hGH epitope according to the present invention may be any fragment whose length is between 6 amino acids and the full length of the hGH polypeptide, preferably a fragment between 6 and 50 amino acids. Fragments carrying the epitope are identified by either the number of consecutive amino acid residues (as a subgroup) or the specific N-terminal and C-terminal positions (as a species) as described above. You can.

[0063] Fragments that function as epitopes may be generated by any conventional means (see, eg, Houghten (1985), Proc. Natl. Acad. Sci. USA 82: 5131-5135 and US Pat. No. 4,631,21). These disclosures are hereby incorporated by reference in their entirety.) Methods for determining the amino acids comprising an epitope include X-ray crystallography, two-dimensional nuclear magnetic resonance, and epitope mapping, such as Geysen et al., (1984), Proc. Natl. Acad. Sci. USA 81: 3998. -4002; including the Pepscan method described by PCT applications WO 84/03564 and WO 84/03506, the disclosures of which are hereby incorporated by reference in their entirety. Another example is the algorithm of Jameson and Wolf, (1988), Comp. Appl. Biosci. 4: 181-186 (the above reference is incorporated by reference in its entirety). The Jameson-Wolf antigen analysis may be performed, for example, using the computer program PROTEAN (Version 4.0 Windows, DNASTAR, Inc.) using default parameters.

  [0064] The invention also excludes any hGH fragment species identified by N- and C-terminal positions as described above, or any fragment of a subgroup identified by the size of amino acid residues. As offered. Any number of fragments specified by N-terminal and C-terminal positions as described above or by the size of amino acid residues may be excluded as individual species. The present invention also provides for excluding any hGH domain or epitope-bearing fragment in a similar manner.

[0065] The hGH polypeptides of the invention can be prepared by any suitable method. Such hGH polypeptides and fragments thereof can be purified from natural materials, chemically synthesized, in vitro translation techniques or hGH using techniques known to those skilled in the art.
These may be produced by recombinant techniques, including expression in recombinant cells capable of expressing cDNA, or a combination of these methods. (For example, “Methods in Enzymology, Academic Press, 1993” for various methods for purifying proteins; Creighton, (1983) Proteins: Structures and Molecular Principles, WH Freeman & Co. 2nd Ed., TE, New York; and Hunkapiller et al., (1984) Nature. 310 (5973): 105-11, and Davis et al. (1986) Basic Methods in Molecular Biology, ed., Elsevier Press, NY See, these disclosures are incorporated by reference in their entirety). The polypeptides of the present invention are preferably provided in an isolated form and may be partially or preferably substantially purified.

[0066] "Polynucleotide having at least x% identity to a reference polynucleotide" and "Polypeptide having at least x% identity to a reference polypeptide"
The term "residues (each nucleotide or amino acid)" is identical or higher than x as compared to the reference polynucleotide or polypeptide sequence, respectively, as defined below. Comprehensively includes a polynucleotide or polypeptide that exhibits a percentage.

[0067] This identity ratio is determined after optimal alignment of the two polynucleotide or polypeptide sequences over the entire comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window is the sequence In order to optimize the alignment, the addition or deletion of one or more residues may be included. The comparison window may contain a number of positions (either residues or gaps corresponding to residue insertion / deletions), the number of positions corresponding to the size of the window. The position of each window may represent one of the following states:

1 ° / There is a residue (nucleotide or amino acid) at this position on the aligned first sequence and a different residue at the same position on the aligned second sequence, in other words the second sequence Has a residue substituted at this position compared to the first sequence.

There is a residue (nucleotide or amino acid) at this position on the 2 ° / aligned first sequence and the same residue at the same position on the aligned second sequence.
3 ° / There is a residue (nucleotide or amino acid) at this position on the aligned first sequence and no residue at the same position on the aligned second sequence, in other words the second sequence Represents a deletion at this position compared to the first sequence.

The number of positions in the comparison window belonging to the first category defined above is called R1.
The number of positions in the comparison window belonging to the second category defined above is called R2.
The number of positions in the comparison window belonging to the third category defined above is called R3.

  [0068] The identity ratio (% id) is calculated using the following formula:

You may calculate by either.
[0069] Alignment of sequences for comparison may be performed using any of a variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs are: TBLASTN, BLASTP, FASTA, TFASTA, FASTDB, WU-BLAST, Gapped-BLAST, PSI-BLAST (Pearson and Lipman, (1988), Proc. Natl. Acad. Sci. USA 85 : 2444-2448; Altschul et
al., (1990), J. Mol. Biol. 215: 403-410; Altschul et al., (1993), Nature hGHtics 3: 266-272; Altschul et al., (1997), Nuc. Acids Res. 25: 3389-3402; Thompson et
al., (1994), Nuc. Acids Res .. 22: 4673-4680; Higgins et al., (1996), Meth. Enzymol. 266: 383-402; Brutlag et al. (1990) Comp. App. Biosci 6: 237-245; Jones and Swindells, (2002) Trends Biochem Sci 27: 161-4; Olsen et al. (1999) Pac Symp Biocomput; 302-13, but in no way limited to these . These disclosures are incorporated by reference in their entirety.

[0070] In certain embodiments, the Smith-Waterman method uses a scoring matrix such as PAM, PAM250, or preferably using a BLOSUM matrix such as BLOSUM60 or BLOSUM62, and default parameters (Gap Opening Penalty = 10 and Use Gap Extension Penalty = 1) or with user specific parameters, preferably better than default parameters.

[0071] In another specific embodiment, the protein and nucleic acid sequences can be generated using the default Local Alignment Search Tool ("BLAST") program using default parameters or using modified parameters provided by the user. Use to align. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., (1992), Science 256: 1443-1445; Henikoff and Henikoff, (1993), Proteins 17: 49-61, the disclosures of which are incorporated herein. Are incorporated by reference in their entirety). Although less preferred, PAM matrices or PAM250 matrices may also be used (eg Schwartz and Dayhoff, (1978), eds., Matrices
for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation, the disclosure of which is hereby incorporated by reference in its entirety.)

[0072] In yet another specific embodiment, polynucleotide and polypeptide sequences are aligned using the FASTDB computer program based on the algorithm of Brutlag et al. (1990) (described above). Preferred parameters used in FASTDB alignment of DNA sequences are as follows: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff
Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 500 or the length of the target nucleotide sequence (whichever is shorter). The preferred parameters used for FASTDB amino acid alignment are as follows: Matrix = PAM 0, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group25Length = 0, Cutoff Score = 1, Window
Size = sequence length, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 or the length of the target nucleotide sequence (whichever is shorter).

  [0073] In accordance with the present invention, poly (ethylene glycol) is covalently linked through the amino acid residues of hGH or agonist variants thereof. A variety of activated poly (ethylene glycols) having several different functional groups, linkers, configurations and molecular weights are known to those skilled in the art and are used to make PEG-hGH conjugates or PEG-hGH agonist variant conjugates. Gates may be made (for review see Roberts MJ et al., Adv. Drug Del. Rev. 54: 459-476, 2002; Harris JM et al., Drug Delivery Systems 40: 538-551, 2001 ). The present invention relates to a method of directing the selectivity of a PEG moiety to the N-terminus using aldehyde chemistry and using a butyrylaldehyde linker moiety. The butyrylaldehyde linker results in increased N-terminal specificity compared to the acetaldehyde linker (Table 1 and FIG. 1).

  [0074] One embodiment of the present invention is a structure of Formula I or Formula II:

[Wherein n is an integer between 1 and 10;
m is an integer between 1 and 10;
R is human growth hormone, methionyl growth hormone, or a human growth hormone variant].
It is a human growth hormone-PEG conjugate having

[0075] In certain embodiments, n is between 1 and 5 and m is between 1 and 5.
[0076] In certain embodiments of formula I: n is 1 and m is 1; n is 1 and m is 2; n is 1 and m is 3; And m is 4; n is 1 and m is 5; n is 1 and m is 6; n is 1 and m is 7; n is 1 , M is 8, n is 1, m is 9, n is 1, m is 10, n is 2, m is 1, n is 2, m Is 2, n is 2, m is 3, n is 2, m is 4, n is 2, m is 5, n is 2, m is 6 N is 2 and m is 7; n is 2 and m is 8; n is 2 and m is 9; n is 2 and m is 10 N is 3 and m is 1; n is 3 and m is 2; n is 3 and m N is 3 and m is 4; n is 3 and m is 5; n is 3 and m is 6; n is 3 and m is 7 N is 3 and m is 8; n is 3 and m is 9; n is 3 and m is 10; n is 4 and m is 1; n is 4 and m is 2; n is 4 and m is 3; n is 4 and m is 4; n is 4 and m is 5; 4 and m is 6; n is 4 and m is 7; n is 4 and m is 8; n is 4 and m is 9; n is 4 M is 10; n is 5; m is 1; n is 5; m is 2; n is 5; m is 3; n is 5; m is 4, n is 5, m is 5, n is 5, m is 6, n is 5 M is 7; n is 5; m is 8; n is 5; m is 9; n is 5; m is 10; n is 6; m is 1, n is 6, m is 2, n is 6, m is 3, n is 6, m is 4, n is 6, m is N is 6 and m is 6; n is 6 and m is 7; n is 6 and m is 8; n is 6 and m is 9 N is 6 and m is 10; n is 7 and m is 1; n is 7
And m is 2, n is 7, m is 3, n is 7, m is 4, n is 7, m is 5, and n is 7. , M is 6, n is 7, m is 7, n is 7, m is 8, n is 7, m is 9, n is 7, m Is 10; n is 8; m is 1; n is 8 and m is 2; n is 8 and m is 3; n is 8 and m is 4 N is 8 and m is 5; n is 8 and m is 6; n is 8 and m is 7; n is 8 and m is 8 N is 8 and m is 9; n is 8 and m is 10; n is 9 and m is 1; n is 9 and m is 2; Is 9 and m is 3; n is 9 and m is 4; n is 9 and m is 5 N is 9 and m is 6; n is 9 and m is 7; n is 9 and m is 8; n is 9 and m is 9; Is 9 and m is 10; n is 10 and m is 1; n is 10 and m is 2; n is 10 and m is 3; n is 10 And m is 4; n is 10 and m is 5; n is 10 and m is 6; n is 10 and m is 7; n is 10 , M is 8, n is 10, m is 9, n is 10, and m is 10.

  [0077] Certain embodiments have the following structure:

[Wherein R is human growth hormone, methionyl human growth hormone, or a human growth hormone variant]
It is a human growth hormone-PEG conjugate having

[0078] Another specific embodiment of the invention is a human growth hormone-PEG conjugate wherein the human growth hormone comprises or consists of the amino acid of SEQ ID NO: 1.
[0079] A specific aspect of the present invention is 80% of polyethylene glycol, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably Is 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%. %, More preferably 95%, more preferably 96%, more preferably 97%, more preferably more than 98% of human growth hormone conjugated with phenylalanine at the amino terminus of the amino acid sequence of SEQ ID NO: 1 PEG conjugate.

[0080] Another specific embodiment of the invention is a human growth hormone-PEG conjugate in which greater than 90% of the polyethylene glycol is conjugated with the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. .

  [0081] Another specific embodiment of the invention is a human growth hormone-PEG conjugate in which greater than 95% of the polyethylene glycol is conjugated with the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. .

  [0082] Another specific embodiment of the present invention is a human growth hormone-PEG conjugate in which more than 98% of the polyethylene glycol is conjugated with the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. .

[0083] The poly (ethylene glycol) used in the present invention is not limited to any particular form or molecular weight range. The molecular weight of poly (ethylene glycol) may be between about 500 and about 100,000 daltons. The term “about” indicates that in the preparation of polyethylene glycol, some molecules are larger than the stated molecular weight, some are smaller, and the stated molecular weight refers to the average molecular weight. It will be appreciated that there is some degree of polydispersity associated with polymers such as poly (ethylene glycol). It is preferable to use PEG with low polydispersity. Typically, PEG with a molecular weight of about 500 to about 60,000 is used. A specific PEG molecular weight range of the present invention is from about 1,000 to about 40,000. In another specific embodiment, the PEG molecular weight is greater than about 5,000 and up to about 40,000. In another specific embodiment, the PEG molecular weight is from about 20,000 to about 40,000. Depending on the desired therapeutic aspect (eg desired duration of release, effect on biological activity, if any, the antigenicity of polyethylene on the therapeutic protein and other known effects or deficiencies) Other sizes may be used. For example, polyethylene glycol has an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10, 000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45, 000, 50,000, 55,000, 60,000, 65,000, 70, 000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Daltons.

[0084] In another embodiment, the poly (ethylene glycol) is a branched PEG having one or more PEG moieties attached (US Pat. No. 5,932,462; US Pat. No. 5,342,940; US Patent 5,643,575; US Patent 5,919,455; US Patent 6,113,906; US Patent 5,183,660; Kodera Y., Bioconjugate Chemistry 5: 283-288 (1994); and WO 02 / 09766). In a preferred embodiment, the molecular weight of each poly (ethylene glycol) in the branched PEG is about 5,000-20,000. In a specific embodiment, the molecular weight of each poly (ethylene glycol) of the branched PEG is about 20,000.

[0085] Poly (alkylene oxide), in particular poly (ethylene glycol), is linked to hGH or an agonist variant thereof via a terminal reactive group, which is a linking moiety (spacer) between PEG and protein. You may or may not leave. In order to form a hGH conjugate of the invention or a conjugate of an agonist variant thereof, a polymer, such as a poly (alkylene oxide), is converted to an active form, such terms are known to those skilled in the art. This reactive group is, for example, a terminal reactive group, which mediates the bond between the chemical moiety of the protein and poly (ethylene glycol). Typically, the hydroxyl terminal groups of one or both terminal polymers (ie, alpha and omega terminal hydroxyl groups) are converted to reactive functional groups that allow covalent conjugation. This process is often referred to as “activation” and the poly (ethylene glycol) product having reactive groups is hereinafter referred to as “activated poly (ethylene glycol)”. In a specific embodiment, one of the terminal hydroxyl-terminated groups of the polymer is transformed or capped with a non-reactive group. In a specific embodiment, the terminal hydroxyl terminal group of the polymer is converted or capped with a methyl group. As used herein, the term “mPEG” refers to PEG capped at one end with a methyl group. mPEG is structurally
CH ₃ O- (CH ₂ CH ₂ O) _n -H
It can be expressed as.

[0086] Polymers containing both α and ε linking groups are referred to as “bis-activated poly (alkylene oxides)” and are referred to as “bifunctional”. Polymers containing the same reactive group at the α and ε terminal hydroxyls are sometimes referred to as “homobifunctional” or “homobis-activated”. Polymers containing different reactive groups at the α- and ε-terminal hydroxyls are sometimes referred to as “heterobifunctional” (see eg WO 01/26692) or “heterobis-
Polymers containing one reactive group are referred to as “mono-activated” polyalkylene oxides or “monofunctional” polyalkylene oxides, as well as other substantially non-antigenic polymers. Similarly, it is “activated” or “functionalized”.

  [0087] Thus, the activated polymer is used to mediate the bond between a chemical moiety of the protein, such as an α- or β-amino, carboxyl, or thiol group, and poly (ethylene glycol). Suitable. The bis-activated polymer reacts in this manner with two protein molecules or one protein molecule, and also with a reactive small molecule in another embodiment, through a cross-linked protein polymer conjugate or protein-small Molecular conjugates can be effectively formed.

[0088] In one preferred embodiment of the present invention, the secondary amine or amide linkage uses an α-amino or ε-amino group of lysine at the N-terminus of hGH or an agonist variant thereof, and an activated PEG. Form. In another preferred aspect of the invention, the secondary amine linkage is a N-terminal primary α-amino or primary ε-amino group of hGH or an agonist variant thereof, and a single chain or branched. A suitable reducing agent, such as Chamow et al., Bioconjugate Chem. 5: 133-140 (1994), US Pat. No. 4,002,531, WO 90/05534, and US Patent No. 5,824,784
Formed by reductive alkylation with NaCNBH ₃ , NaBH ₃ , pyridine borane, etc.

  [0089] In a preferred embodiment, at least 70%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94% , Preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% of the poly (ethylene glycol) is present in the amino terminal α-amino group.

[0090] Conjugation reactions, also referred to as pegylation reactions, have historically been carried out in solution with an excess molar amount of polymer, regardless of where the polymer binds to the protein. However, such general techniques have proven to be typically unsuitable for conjugating bioactive proteins with non-antigenic polymers while retaining sufficient bioactivity. One method for maintaining the biological activity of hGH or an agonist variant thereof is a reactive group involved in the receptor binding site (s) of hGH or an agonist variant thereof in a method of coupling with a polymer. The conjugation at is substantially avoided. Another aspect of the invention is to provide a method of conjugating poly (ethylene glycol) with hGH or agonist variants thereof while maintaining a high level of retained activity.

[0091] Chemical modification through covalent bonds may be performed under appropriate conditions generally accepted in the reaction of biologically active substances with activated poly (ethylene glycol). The conjugation reaction is performed under relatively mild conditions to avoid inactivating hGH or agonist variants thereof. Mild conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperature in the range of about 0 ° -37 ° C. If the reactive amino acid residue in hGH or an agonist variant thereof has a free amino group, the above modification is preferably performed with phosphate, MES, citric acid, acetic acid, succinic acid, or HEPES buffers. In a non-limiting list of suitable buffers (pH 3 to 10), including 1-48 hours at 4 ° -37 ° C. When targeting the N-terminal amino group with a reagent such as PEG aldehyde, it is preferably maintained at pH 4-7. Activated poly (ethylene glycol) is used at about 0.01-100 times, preferably about 0.01-2.5 times the molar amount of the number of free amino groups in hGH or agonist variants thereof. You can. In contrast, if the reactive amino acid residue in hGH or an agonist variant thereof has a free carboxyl group, the above-described modification is preferably performed at a pH of about 3.5 to about 5.5; For example, modification with poly (oxyethylenediamine) is carried out at 4 ° -37 ° C. for 1-24 hours in the presence of carbodiimide (pH 4-5). Activated poly (ethylene glycol) may be used at 0.01-300 times the molar amount of the number of free carboxyl groups of hGH or agonist variants thereof.

  [0092] In an independent embodiment, the upper limit of the amount of polymer included in the conjugation reaction is about 1 per molecule of hGH or an agonist variant thereof without forming a substantial amount of high molecular weight species. More than about 1: 1 to the extent that the activated polymer can be reacted with hGH or an agonist variant thereof without more than about 20% conjugate containing more polymer chains than this. For example, in this aspect of the invention, ratios up to about 6: 1 can be used to form significant amounts of the desired conjugate that can be subsequently isolated from any high molecular weight species. Conceivable.

[0093] In another aspect of the invention, a polymeric hGH or agonist thereof, wherein a bifunctional activated PEG derivative is used to cross-link multiple hGH or agonist variant molecules via PEG Variant-PEG molecules may be produced. While the reaction conditions described herein will result in significant amounts of unmodified hGH or agonist variants thereof, unmodified hGH or agonist variants thereof may be used for further conjugation reactions. Therefore, it can be easily reused in future batches. The methods of the present invention surprisingly few, i.e., less than about 30%, high molecular weight species, and species (s) containing more than one polymer chain per molecule of hGH or agonist variants thereof. And more preferably produces less than about 10%. These reaction conditions are in contrast to the conditions typically used for polymer conjugation reactions where the activated polymer is present in a several fold molar excess over the target. In other aspects of the invention, the polymer is present in an amount of about 0.1 to about 50 equivalents per equivalent of hGH or agonist variant thereof. In another aspect of the invention, the polymer is present in an amount of about 1 to about 10 equivalents per equivalent of hGH or agonist variant thereof.

[0094] The conjugation reactions of the present invention involve mono-PEG-hGH conjugates and di-PEG-hGH conjugates, unreacted hGH, unreacted polymers, and typically high molecular weight species less than about 20%. The reaction mixture or pool containing is first provided. The high molecular weight species is a conjugate containing one or more polymer chains, and / or
Or a conjugate containing a species of polymerized PEG-hGH or agonist variant thereof. After removal of unreacted species and high molecular weight species, the composition containing primarily conjugates of mono-polymer and di-polymer-hGH or agonist variants thereof is recovered. Considering the fact that most conjugates contain one polymer chain, this conjugate is substantially uniform. These modified hGH or agonist variants thereof are standard FDC-P1 cell proliferation assays (Clark et al. Journal of Biological Chemistry 271: 21969-21977, 1996), receptor binding assays (US Pat. No. 5,057). 417) or with hypophysectomized rat growth (Clark et al. Journal of Biological Chemistry 271: 21969-21977, 1996), in association with native or unmodified hGH or agonist variants thereof. have at least about 0.1% of the biological activity in vitro. However, in a preferred aspect of the invention, the modified hGH or agonist variant thereof has about 25% of the in vitro biological activity, more preferably the modified hGH or agonist variant thereof is an in vitro organism. More preferably, the modified hGH or agonist variant thereof having about 50% of the biological activity has about 75% of in vitro biological activity, and most preferably the modified hGH or agonist variant thereof Have equal or improved in vitro biological activity.

  [0095] The methods of the present invention preferably comprise rather limited ratios of polymer to hGH or agonist variants thereof. Thus, it has been discovered that conjugates of hGH or agonist variants thereof are mainly limited to species containing only one polymer chain. Furthermore, the attachment of the polymer to the reactive group of hGH or an agonist variant thereof is substantially less random than when using a higher molar excess of polymer linker. Unmodified hGH or agonist variants thereof present in the reaction pool can be used for subsequent reactions using ion exchange chromatography or size exclusion chromatography or similar separation techniques after stopping the conjugation reaction. Can be used.

[0096] Poly (ethylene glycol) modified hGH or agonist variants thereof, ie proteins chemically modified according to the present invention, can be prepared using conventional methods such as dialysis, salting out, ultrafiltration, The reaction mixture may be purified by ion exchange chromatography, hydrophilic interaction chromatography (HIC), gel chromatography, and electrophoresis. Ion exchange chromatography is particularly effective in removing unreacted poly (ethylene glycol) and hGH or agonist variants thereof. In a further aspect of the invention, mono-polymer and di-polymer-hGH or agonist variants thereof are isolated from the reaction mixture to remove high molecular weight species and unmodified hGH or agonist variants thereof. . Separation can be achieved by applying mixed species in a buffer containing about 0.5-10 mg / mL hGH or agonist variant-polymer conjugate thereof to a column. Suitable solutions have a pH of about 4 to about 10. This solution is preferably one selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , CH ₃ CO ₂ H, and NaOH. Or more buffer salts.

[0097] Depending on the reaction buffer, the solution of the conjugate of hGH or an agonist variant thereof and the polymer must first be buffer exchanged / ultrafiltered to remove any unreacted polymer. For example, a conjugate solution of PEG-hGH or an agonist variant thereof can be ultrafiltered through a low molecular weight cut-off (10,000 to 30,000 daltons) membrane to remove most undesirable materials such as unreacted polymers. If it is contained, the surfactant and the like can be removed.

  [0098] Fractionation of the conjugate into a pool containing the desired species is preferably performed using ion exchange chromatography media. Such media can selectively bind conjugates of PEG-hGH or agonist variants thereof, with charge differences that change to some degree in a predictable manner. For example, the surface charge of hGH or an agonist variant thereof is determined by the number of available charged groups on the surface of the protein. These charged groups are typically the points at which poly (alkylene oxide) polymers can be attached. Thus, conjugates of hGH or agonist variants thereof will have a different charge than other species that allow selective isolation.

[0099] Strongly polar anion or cation exchange resins, such as quaternary amine resins or sulfopropyl resins, respectively, are used for the process of the present invention. Ion exchange resins are particularly preferred. A non-limiting list including commercially available cation exchange resins suitable for use in the present invention is SP-hitrap®, SP Sepharose HP®, and SP Sepharose® fast flow. (High flow rate). Other suitable cation exchange resins such as S and CM resins can also be used. A non-limiting list of anion exchange resins, including commercially available anion exchange resins suitable for use in the present invention, is Q-hitrap®, Q Sepharose HP®, and Q sepharos®. Trademark) fast flow. Other suitable anion exchange resins such as DEAE resins can also be used.

[00100] For example, an anion or cation exchange resin is preferably packed into a column and equilibrated by conventional means. A buffer having a pH and osmolality equal to the solution of hGH or agonist variant thereof conjugated to the polymer is used. The elution buffer is preferably one selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , and (NH ₄ ) ₂ CO _3. Or it contains more salt. The solution containing the conjugate is then adsorbed onto the column, along with unreacted polymer and some high molecular weight species not retained on the column. Once the sample is loaded on the column, elution buffer gradient with increasing salt concentration is performed to elute the desired fraction of hGH or its agonist variant conjugated with polyalkylene oxide. The eluted and pooled fractions are preferably limited to uniform polymer conjugates after the cation or anion exchange resin step. Any unconjugated hGH or agonist variant thereof can then be backwashed from the column by conventional techniques. If desired, mono- and multi-pegylated hGH or agonist variant species thereof can be further separated from each other by further ion exchange chromatography or size exclusion chromatography.

  [00101] Techniques that utilize multiple isocratic steps while increasing salt concentration or pH can also be used. Multiple homogeneous concentration elution steps with increasing concentrations will result in sequential elution of di- and then mono-hGH or agonist variant-polymer conjugates thereof.

[00102] The temperature range for elution is between about 4 ° C and about 25 ° C. Preferably, elution is performed at a temperature from about 4 ° C to about 22 ° C. For example, elution of a fraction of PEG-hGH or an agonist variant thereof is detected by UV absorbance at 280 nm. Collection of fractions may be achieved with a simple time elution profile.

[00103] Surfactants can be used in methods of conjugating poly (ethylene glycol) polymers with portions of hGH or agonist variants thereof. Suitable surfactants include ionic type materials such as sodium dodecyl sulfate (SDS). Other ionic surfactants such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, etc. can also be used. Nonionic surfactants can also be used. For example, materials such as poly (oxyethylene) sorbitan (Tweens) and poly (oxyethylene) ether (Tritons) can be used. See also Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Corp. The only limitation of the surfactant used in the method of the present invention is that it does not cause substantially irreversible denaturation of hGH or agonist variants thereof and does not completely inhibit polymer conjugation. Use them under and at concentrations. The surfactant is present in the reaction mixture in an amount of about 0.01-0.5%; preferably 0.05-0.5%, and most preferably about 0.075-0.25%. And A mixture of surfactants may also be considered.

[00104] Surfactants are believed to provide a temporary, reversible protection system during the method of polymer conjugation. Surfactants have been shown to be effective in selectively preventing polymer conjugation while allowing lysine-based or amino-terminal based conjugation to be facilitated.

[00105] The poly (ethylene glycol) modified hGH or agonist variants thereof of the present invention have a more sustained pharmacological effect, which is likely due to an extended half-life in vivo. .

[00106] Another aspect of the invention is a poly (ethylene glycol) modified h of the invention
Diseases in which the use of GH, preferably hGH, is beneficial, comprising administering to a patient in need thereof a therapeutically effective amount of GH or an agonist variant thereof, alone or in combination with another therapeutic agent Or relates to a method for the prevention and / or treatment of disorders. The present invention also provides
It relates to the use of a poly (ethylene glycol) modified hGH of the invention or an agonist variant thereof in the manufacture of a pharmaceutical agent for the prevention and / or treatment of diseases or disorders where the use of GH, preferably hGH, is beneficial. In addition, the present invention also encompasses the poly (ethylene glycol) modified hGH of the present invention or agonist variants thereof for the prevention and / or treatment of diseases or disorders where the use of GH, preferably hGH, is beneficial. To a pharmaceutical composition.

[00107] Diseases or disorders where the use of GH is beneficial are growth hormone deficiency (GHD)
), Adult growth hormone deficiency (aGHD), Turner syndrome, growth failure in children born short of gestational age (SGA), Praderwilly syndrome (PWS), chronic renal failure (CRI), AIDS Wasting, aging, end-stage renal failure, cystic fibrosis, erectile dysfunction, HIV lipodystrophy, fibromyalgia, osteoporosis, memory impairment, depression, Crohn's disease, skeletal dysplasia, traumatic brain Injury, subarachnoid hemorrhage, Noonan syndrome, Down syndrome, idiopathic short stature (ISS), end stage renal disease (ESRD), very low birth weight infant (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, Including, but not limited to, short stature resulting from glucocorticoid treatment in children, and premature infant short stature catch-up growth failure.

[00108] In a more specific embodiment of the present invention, the poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention is selected from the group consisting of GHD, aGHD, SGA, PWS, Turner syndrome and CRI. Used in the prevention and / or treatment of certain disorders or diseases.

[00109] In another more specific embodiment of the present invention, the poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention is treated with idiopathic short stature, very low birth weight infant, traumatic brain. Use in the prevention and / or treatment of a disorder or disease selected from the group consisting of injury, metabolic syndrome, and Noonan syndrome.

[00110] Another aspect of the invention is a poly (ethylene glycol) modified h of the invention
It relates to a pharmaceutical composition comprising GH or an agonist variant thereof alone or in combination with another therapeutic agent and at least one pharmaceutically acceptable excipient or carrier. The poly (ethylene glycol) modified hGH or agonist variants thereof of the present invention can then be pharmaceutically acceptable diluents, substances for preparing isotonic solutions, pH for administration to patients, pH You may formulate in the medical chemical | medical agent containing a modifier.

[00111] The above drugs may be subcutaneous, intramuscular, intravenous, transpulmonary, intradermal, depending on the purpose of the treatment,
Or it may be administered orally. The dose may also be based on the type and condition of the disorder being treated, but is typically between 0.1 m and 5 mg by injection for adults and between 0.1 mg and 50 mg for oral administration.

[00112] As used herein, the poly (ethylene glycol) modified hG of the present invention
H or an agonist variant thereof may be used in combination with another therapeutic agent. As used herein, the terms “in combination”, “in combination” and “in combination with” with respect to Compound A and one or more other therapeutic agents mean: Exactly mentioned and intended to include:
-Co-administration of such a combination of A and therapeutic agent (s) to a patient in need of treatment, where such ingredients are combined in one dosage form from which Ingredients are released to the patient at substantially the same time.

-Substantial simultaneous administration of such a combination of A and therapeutic agent (s) to a patient in need of treatment, where such components are separately taken by said patient at substantially the same time In which the ingredients are released to the patient at substantially the same time-such as A and therapeutic agent (s) to the patient in need of treatment Continuous administration in such a combination, in which case such ingredients are formulated separately from each other in separate dosage forms that are taken by said patient at successive times with a significant time interval between each administration, in which case Said component is released to said patient at substantially different times; and-such administration of such combination of A and therapeutic agent (s) to a patient in need of treatment, in this case such component The control Combined into one dosage form that releases the components in a controlled manner, wherein these components are simultaneously, sequentially, and / or overlapping by the patient at the same and / or different times Be administered.

[00113] Suitable examples of other therapeutic agents that may be used in combination with A, their pharmaceutically acceptable salts, and / or their derivatives include, but are in no way limited to: Not: aromatase inhibitors such as exemestane, formestane, atamestan, fadrozole, letrozole, borozole, and anastrozole; fibric acid derivatives (eg fenofibrate, clofibrate, gemifibrozil, bezacibrate and ciprofibrate) and nicotine Acid derivatives eg fatty acid modulators including acipimox; biguanides eg insulin resistance ameliorators including but not limited to metformin, PPAR gamma insulin sensitizers and thiazolodeniones eg troglitazone Rosiglitazone, troglitazone, 5-[[4- [3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-]-benzopyran-2-yl) methoxy] phenyl] methyl 3 -2,4-thiazolidige screw V
411 (DIABII, Glaucanin), Pigoritazone (ACTOS, AD 4833, U 72107, U 72107A, U 72107E, ZACTOS) Chemical names: 2,4-thiazolidinedione, 5-[[4- [2- (5-ethyl) -2-pyridyl) ethoxy] phenyl] methyl]-, monohydrochloride, (a /-); rosiglitazone (Avandia, BRL 49653, BRL 4
9653C) Chemical name: 2,4 thiazolidinone, 5-[[4- [2- (methyl-2-pyridinylamino) ethoxy] phenyl] methyl]; 25 bexarotene-oral (LGD 1069 oral, tarretin oral) , Targretin, targretin (oral), chemical name: 4- [1- (3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) Ethyl] benzoic acid; ZD 2079, (I
CI D 2079) (Chemical name: R) -N- [2-4- (carboxymethyl) 30phenoxy] ethyl) -N- (2-hydroxy-2-phenethyl) ammonium chloride: Netoglitazone, (Isaglitazone) , MCC 555, RWJ 241947) (Chemical name: 5- [6 (2-Fluorobenzyloxy) naphthalen-2-ylmethyl] thiazolidine-2,4
-Dione); INS (D-kilo-inositol) (chemical name: D-1,2,3,4,5,6-hexahydroxycyclohexane), ON 2344 (DRF 2593); dexlipotam, chemical name : 5 (R)-(1,2-dithiolan-3-yl) pentaloic 35 acid; HQL 975, chemical name: 3- [4- [2- (5-methyl-2-phenyloxazol-4-yl) Ethoxy] phenyl] -2 (S)-(propiolamino) propionic acid; YM 268, chemical name: 5,5′-methylene-bis (1,4-phenylene) bismethylenebis (thiazolidine-
2,4-dione). IPPAR agonists under development include: Reglitars (JTT
501, PNU 182716, PNU 716) (chemical names: isoxazolidien-3,5-dione, i 4-[[4- (2-phenyl-5-methyl) -1,3-oxazolyl] ethoxyphenyl-4 ] Methyl-, (4RS)); I (RP 297, chemical name: 10 5- (2,4-
Dioxothiazolidine-5-ylmethyl) -2-methoxy-N- [4- (trifluoromethyl) benzylbenzamide; R 119702 (CI 1037, CS 011) Chemical name: (/-)-5- [4- (5 -Methoxy-1H benzimidazol-2-ylmethoxy) benzyl] thiazoline-2,4-dione; hydrochloride salt; 15 DRF 2189, chemical name: 5-[[4- [2- (1-indolyl) ethoxy] phenyl] Methyl] thiazolidine-2,4-diones; cortisol synthesis inhibitors such as ketoconazole, econazole or miconazole; growth hormones such as somatropin or somatonorm, and derivatives thereof such as human growth hormone fusion proteins such as ALBUTROPIN; polyethylene glycol growth hormone such as cysteine -Pegylated growth hormone, B 005 (Bolder BioTechnology Inc.); growth hormone secretagogues such as SM 130686 (Sumitomo), capromorelin (Pfizer), mecasermin (Fujisawa), sermorelin (Salk Institute, Bio-Technology General), somatrem, somatomedin (C Llorente; Pharmacia Corporation), Examorelin, tabimorelin; CP 464709 (Pfizer), LY 426410 and LY 444711 (Lilly); 8- (aminoalkoxyimino) -8H-dibenzo as disclosed in WO2002057241 [a, e] triazolo [4,5-c] cycloheptenes, 2-substituted dibenzo [a, e] 1,2,3-triazolo [4,5-c] [7] as disclosed in WO2002056873 Anulen-8-ones, U.S. Pat. No. 4,411,890 and public information WO 89/07110, W Growth hormone releasing peptides GHRP-6 and GHRP-1 as described in 89/07111, B-HT920, hexarelin and GHRP-2 as described in WO 93/04081, or growth hormone releasing hormone (GHRH) Or so-called GRF) and analogs thereof, somatomedins including IGF-1 and IGF-2, and derivatives thereof, such as recombinant fusions of somatocaine-ie insulin-like growth factor-1 and its binding protein BP-3, Alpha-2-adrenergic agonists such as clonidine, xylazine, detomidine and medetomidine, or seretonin 5HTID agonists such as sulnitriptan, or somatostatin or substances that inhibit its release Example, if physostigmine and pyridostigmine, Th
GRF 1-44 (Theratechnologies); L 165166 (Merck &Company); dipeptide derivatives as described in WO 9858947, inhibitors of dipeptidyl peptidase IV eg as described in US 6521644, WO 95/15309 and WO 98/19998 Amino-acylpyrrolidine nitriles; beta-amino heterocyclic dipeptidyl peptidase inhibitors such as those described in US200330100563 and WO2003082817; US20030055261, US2003004
0483, EP 18 072, EP 83 864, WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272,
WO 91/18016, WO 92/01711, WO 93/04081, WO 95
14666, EP0923539, U.S. Pat.Nos. 5,206,235, 5,283,241, 5,284,841, 5,310,737, 5,317,017, 5,374,721, 5 , 430, 144, 5,434, 261, 5,438, 136, 5,494, 919, 5,494, 920, 5,492, 916, 5, 536, 7
16, and 5,578,593, WO 94/13696, WO 94/19367, WO 95/03289, WO 95/03290, WO 95/09633, WO 9
5/11029, WO 95/12598, WO 95/13069, WO 95/14666, WO 95/16675, WO 95/16692, WO 95/17422, WO
95/17423, growth hormone releasing compounds as described in WO 95/34311, and WO 96/02530, piperidine, pyrrolidine and hexahydro-1H-adipine, amidospiro as described in US 5804578, US 5578582, WO 2004007468 Piperidines, such as those described in WO010104119, 2-[(5,6-dimethyl-2-benzimidazolyl) amino] -4-hydroxy-6-methyl-5-pyrimidineacetic acid (2 ) And 2-[(5,6-Dimethyl-2-benzimidazolyl) amino] -4-hydroxy-6-methyl-5-pyrimidineacetic acid, 2-amino-5-pyrimidineacetic acid compounds including ethyl ester, described in EP 1155014 I have Benzimidazole, peptidyl compound analogs and peptides for GRF of US Pat. 6-azaindole compounds as described in WO 9921553, and in WO0053602, WO0053185, WO0053181, WO0053180, WO0053179, WO0053178, US62888078; IGF-1 secretagogue; insulin-like growth factor-2 (IGF-2 or somatomedy A) and IGF-2 secretagogues; myostatin antagonist and fibroblast growth factor receptor -3 (FGFR-3) compounds that inhibit tyrosine kinases, and the like.

[00114] The polymeric material included is also preferably water soluble at room temperature. A non-limiting list of such polymers includes poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycol), poly (oxyethylated polyol),
These copolymers and their block copolymers are included, provided that the water solubility of the block copolymers is maintained.

[00115] As an alternative to polymers based on PEG, practically non-antigenic materials such as dextran, poly (vinyl pyrrolidone), poly (acrylamide), poly (vinyl alcohol), hydrocarbon based polymers, etc. Can be used. In fact, activation of the α- and ε-terminal groups of these polymeric materials can be accomplished in a manner similar to that used to convert the poly (alkylene oxide) and is therefore apparent to those skilled in the art. Will. One skilled in the art will appreciate that the foregoing list is merely illustrative and that all polymer materials having the attributes described herein are contemplated. For the purposes of the present invention, “virtually non-antigenic” is any material that is non-toxic in mammals and understood in the art as not causing any appreciable immunogenic response. Means.

(Definition)
[00116] The following is a list of abbreviations and their corresponding meanings when used with interchangeable surnames herein:
g Gram (s)
mg milligram (s)
ml or ml milliliter (s)
RT Room temperature PEG Poly (ethylene glycol)
[00117] The entire contents of all public relations, patents, and patent applications cited in this disclosure are specifically and individually indicated for each individual public relations, patent, and patent application to be incorporated by reference. And incorporated herein by reference.

[00118] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be appreciated that changes and modifications can be made without departing from the spirit and scope of the invention. In view of the intent of the present invention, it will be readily understood by those skilled in the art. The following examples are provided for illustrative purposes only, and are not intended to limit the scope of the invention, which is described in broad terms above.

[00119] In the following examples, hGH is that of SEQ ID NO: 1. It is understood that other members of the polypeptide family of hGH or agonist variants thereof can also be PEGylated in a manner similar to that illustrated in the Examples below.

(Example 1)
(Branched 40,000 MW PEG-butyraldehyde hGH)

[00120] This example demonstrates a method for the production of a substantially uniform preparation of hGH mono-pegylated at the N-terminus by reductive alkylation. An approximately 40,000 MW methoxy-branched PEG-butyraldehyde reagent (Shearwater Corp.) is used to determine the pK _a value of the N-terminal primary amine versus the pK of the primary amine at the ε-amino position of the lysine residue. _By using the relative difference in _a values, they were selectively coupled via reductive amination of the N-terminus of hGH. 25 mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0, (25 m if desired
The hGH protein dissolved in MMES (Sigma Chemical, St. Louis, MO) pH 6.0, 10 mM sodium acetate (Sigma Chemical, St. Louis, MO) pH 4.5) at 10 mg / mL was methoxy-PEG-butyraldehyde. And M-PEG-ALD (Shear
water Corp., Huntsville, AL) was added to react with M-PEG-ALD to give a relative PEG: hGH molar ratio of 2: 1. The reaction is stock 1M dissolved in H ₂ O.
The catalyst was made by adding NaCNBH ₄ (Sigma Chemical, St. Louis, MO) to a final concentration of 10-50 mM. The reaction was carried out in the dark at room temperature for 18-24 hours. Reactions were added by adding 1 M Tris (Sigma Chemical, St. Louis, Mo.) to pH 7.6 to a final Tris concentration of 50 mM, or by diluting in an appropriate buffer for immediate purification. Stopped.

[00121] Table 1 shows 40K branched PE as determined by size exclusion chromatography.
Shows multiple PEGylated species, mono-pegylated conjugate, percent unreacted PEG, and final product yield for G-aldehyde and 40K branched PEG-butyraldehyde. PEG-butyraldehyde results in increased monopegylated conjugates, decreased levels of unreacted PEG, and increased final yield compared to PEG-aldehyde.

(Example 2)
(Linear 30,000 MW PEG-butyrylaldehyde hGH)
[00122] A methoxy-linear 30,000 MW PEG-butyrylaldehyde reagent is coupled to the N-terminus of hGH using the method described in Example 1.

(Example 3)
(Linear 20,000 MW PEG-butyrylaldehyde hGH)
[00123] A methoxy-linear 20,000 MW PEG-butyrylaldehyde reagent is coupled to the N-terminus of hGH using the method described in Example 1.

Example 4
(Pegylated hGH purification)
[00124] PEGylated hGH species were purified to> 95% (SEC analysis) from the reaction mixture using a single ion exchange chromatography step.

(Anion exchange chromatography)
[00125] Pegylated hGH species were purified to> 95% (SEC analysis) from the reaction mixture using a single anion exchange chromatography step. Mono-pegylated hGH was purified from unmodified hGH and multi-pegylated hGH species using anion exchange chromatography. A typical 20K butyrylaldehyde hGH reaction mixture (5-100 mg protein) was equilibrated with 25 mM HEPES, pH 7.3 (buffer A), as described above, in a Q-Sepharose Hitrap column (1 or 5 mL) (Amersham Pharmacia Biotech, Piscataway, NJ) or Q-Sepharose fast flow column (26/20, 70 m
L bed volume) (Amersham Pharmacia Biotech, Piscataway, NJ). The reaction mixture was diluted 5-10 times with buffer A and applied to the column at a flow rate of 2.5 mL / min. The column was washed with 8 times the column volume of buffer A. The various hGH species were then eluted with a concentration gradient of 80-100 column volumes of buffer A and 0-100 mM linear NaCl. The eluate was monitored by absorbance at ₂₈₀ nm (A ₂₈₀ ) and collected in 5 mL fractions. Fractions were pooled as the degree of pegylation (as assessed in Example 15), eg mono, di, tri, etc. The pool solution was then concentrated to 0.5-5 mg / mL with a Centriprep YM10 concentrator (Amicon, Technology Corporation, Northborough, Mass.). The protein concentration of the pooled solution was determined using an extinction coefficient of 0.78 by _{A 280.}

(Cation exchange chromatography)
[00126] Cation exchange chromatography was performed on a SP Sepharose high performance column (Pharmacia XK 26/20, 70 ml bed volume) equilibrated with 10 mM sodium acetate pH 4.0 (buffer B). . The reaction mixture was diluted 10-fold with buffer B and applied to the column at a flow rate of 5 mL / min. Next, it was washed with 5 times the column volume of buffer B, followed by 5 times the column volume of 12% buffer C (10 mM acetate buffer pH 4.5, 1 M NaCl). The PEG-hGH species was then eluted from the column with a linear concentration gradient of 12 to 27% buffer C, 20 times the column volume. The eluate was monitored at 280 nm and collected in 10 mL fractions. Fractions were pooled according to the degree of pegylation (mono, di, tri, etc.), exchanged for 10 mM acetate pH 4.5 buffer and concentrated to 1-5 mg / mL in a stirred cell set with Amicon YM10 membrane. . Protein concentration of pool solution is determined using an extinction coefficient of 0.78 by _{A 280.}

(Example 5)
(Biochemical characterization)
[00127] The purified pegylated hGH pool solution was characterized by non-reducing SDS-PAGE, non-denaturing size exclusion chromatography, and peptide mapping.

(Size Exclusion High Performance Liquid Chromatography) (SEC-HPLC)
(Non-denaturing SEC-HPLC)
[00128] Reaction of methoxy-PEG of various conjugation chemistries, sizes, linkers and geometries with hGH, purified pool solution by anion exchange chromatography, and final purified product using non-denaturing SEC-HPLC And evaluated. Analytical non-denaturing SEC-HPLC was performed using a column, Superdex 200 7.8 mm x 30 cm (Amersham Bioscience, Piscataway, NJ), in 20 mM phosphate buffer pH 7.2, 150 mM NaCl, at a flow rate of 0.5 mL. Per minute (optionally Tosohaas G4000PWXL Amersham Bioscience, Piscataway, NJ). PEGylation greatly increases the hydrodynamic volume of the protein leading to a shift to a faster retention time. New species were observed in the PEG aldehyde hGH reaction mixture along with unmodified hGH. These pegylated and non-pegylated species are separated on Q-Sepharose chromatography and the resulting purified mono-P
The EG-aldehyde hGH species was subsequently shown to elute as a single peak with non-denaturing SEC (> 95% purity). The Q-Sepharose chromatography step effectively removed free PEG, hGH, and multiply PEGylated hGH species from mono-PEGylated hGH.

(Denaturing SEC-HPLC)
[00129] Reaction of butyrylaldehyde polyethylene glycol with hGH, purification by anion exchange chromatography, and final purified product are evaluated using denaturing SEC-HPLC. Analytical denaturing SEC-HPLC was performed using a Tosohaas 3000 SWXL column 7.8 mm x 30 cm (Tosohaas Pharmacia Biotech, Piscataway, NJ) in 100 mM phosphate buffer pH 6.8, 0.1% SDS at a flow rate of 0.8 mL. Went at / min. PEGylation greatly increases the hydrodynamic volume of the protein and changes it to a faster retention time. PEGylated and non-PEGylated species are separated on Q-Sepharose chromatography.

(SDS PAGE / PVDF transcription)
[00130] Reaction of PEG butyrylaldehyde with hGH using SDS-PAGE,
And the purified final product was evaluated. SDS-PAGE is performed on reducing and non-reducing conditions on 1-20 mm thick 10-20% Tris tricine gel (Invitrogen, Carlsbad, Calif.) And Novex Colloidal Coomassie® Staining was performed using the G-250 staining kit (Invitrogen, Carlsbad, CA). Bands are blotted onto PVDF membrane for subsequent N-terminal sequence identification.

(Analytical anion exchange HPLC)
[00131] The PEG butyrylaldehyde / hGH reaction mixture, purified fractions by anion exchange chromatography, and the final purified product were evaluated using analytical anion exchange HPLC. Analytical anion exchange HPLC was performed using a Tosohaas Q5PW or DEAE-PW anion exchange column, 7.5 mm x 75 mm (Tosohaas Pharmacia Biotech, Piscataway, NJ) at a flow rate of 1 mL in 50 mM Tris ph 8.6. Went at / min. Samples were eluted with a linear gradient of 5-200 mM NaCl.

(N-terminal sequence and peptide mapping)
[00132] using automated Edman degradation chemistry, NH 2 _- to determine the protein sequence of the terminal. An Applied Biosystems Model 494 Procise sequencer (Perkin Elmer, Wellesley, Mass.) Was used for the degradation. Each RTH-AA derivative is Perkin Elmer / Brownlee 2.1 mm ID PT
Identified by on-line RP-HPLC analysis using an Applied Biosystems Model 140C PTH analyzer connected to an H-C18 column.

[00133] Trypsin digestion was performed at a concentration of 1 mg / mL material, typically 50 μg of material was used for one digestion. Trypsin was added so that the ratio of trypsin to PEG-hGH was 1:30 (w / w). Tris buffer is 30 mM, pH 7.5.
Samples were incubated at room temperature for 16 ± 0.5 hours. The reaction was stopped by adding 50 μL of 1N HCl per mL of digestion solution. Prior to placing the sample on the automated sample meter, the sample was diluted in 6.25% acetonitrile to a final concentration of 0.25 mg / ml. Acetonitrile was added first (to 19.8% acetonitrile) and after gentle mixing, water was added to a final volume (4 times the starting volume). Excess digestion solution may be removed and stored at -20 ° C for up to 1 week.

  [00134] Although the Waters Alliance 2695 HPLC system was used for the analysis, other systems should give similar results. The column used was a 25 cm × 4.6 mm column of 5 μm particle size Astec C-4 polymer. The experiment was performed at ambient temperature, typically 50 μg of protein per sample per column. Buffer A is 0.1% trifluoroacetic acid in water; Buffer B is 0.085% trifluoroacetic acid in acetonitrile. The slope was as follows:

[00135] The column is heated to 40 0 C using a heat jacket. Peaks were detected using a Waters 996 PDA detector and data between 210 and 300 nm was collected. The resulting 214 nm chromatogram was used for sample analysis.

[00136] Mapping with trypsin was performed using hGH, 40K branched PEG-aldehyde,
And 40K branched PEG-butyrylaldehyde (FIG. 1). The N-terminal trypsin fragment was designated T-1. The percentage of T-1 present compared to non-pegylated hGH is shown in Table 2. This data shows that when using PEG-aldehyde, 90% of the PEG modification is at the N-terminus, with the rest apparently compared to more than 98% PEG modification at the N-terminus by using PEG-butyrylaldehyde. Suggesting that it is linked to one of several possible lysine residues.

(Example 6)
(Pharmacokinetic study)
(Rat weight gain)
[00137] Female Sprague Dawley rats hypophysectomized at Taconic Labs were pre-screened for growth rate for 7 to 11 days. Rats were divided into 8 groups. One group consists of rats given vehicle subcutaneously daily or on either day 0 and day 6. Group 2 received daily subcutaneous administration of GH (30 μg / rat / dose). Group 3 received GH (180 μg / rat / dose) subcutaneously on day 0 and day 6. Group 4 received 40 k branched PEG-butyrylaldehyde hGH (180 μg / rat / dose) subcutaneously on day 0 and day 6. Hypophysectomized rats were monitored for weight gain by weighing at least every other day during the study. 3 and 4.

(Length of rat tibia)
[00138] Animals from the 11-day weight gain study were sacrificed on day 11, the left tibia was excised, X-rayed, and bone length was measured using calipers. FIG.

(IGF-1 test)
[00139] Animals from a 6 day weight gain study were used. Blood samples were drawn at various times during the study and serum IGF-1 levels were determined by ELISA. FIG.

(Serum biochemistry test)
[00140] Serum biochemistry values were obtained on day 7 after cumulative administration of 1.8 mg / kg on day 0 and day 6, as shown in Table 3, using animals from the 11 day weight gain study. evaluated.

[00141] Day 11 blood urea nitrogen concentrations were significantly (p <0.10) decreased in hGH-treated and PHA-794428-treated animals to the same extent as compared to the vehicle group.
This indicates increased nitrogen utilization as a result of new protein synthesis during enhanced growth.

(Pharmacokinetic study)
[00142] Pharmacokinetic studies were performed in normal, catheterized male Sprague Dawley rats. Injections were performed as a single subcutaneous bolus dose of 100 μg / kg / rat GH or PEG-GH, with 6 rats per group. Blood samples were collected over 1 to 5 days as appropriate for evaluation of relevant PK parameters. Blood levels of GH and PEG-GH were monitored by immunoassay for each sampling.

(HGH immunoassay)
[00143] Protein concentration levels of hGH and PEGylated hGH in mouse and cynomolgus monkey plasma were determined using the hGH AutoDELFIA kit fluorescence immunoassay (PerkinElmer). Rat and human IGF-1 levels were monitored by an immunoassay kit (Diagnostic System Laboratories).

Non-compartmental pharmacokinetic properties for the hGH-PEG conjugate of Example 1 in non-human primates
[00144] The hGH-PEG conjugate of Example 1 was administered to cynomolgus monkeys as a 0.18 mg / kg intravenous (iv) or subcutaneous (sc) bolus injection (Table 4). PK parameters were determined using the average of data from n = 3 animals. Plasma concentrations were measured using the AutoDELFIA kit fluorescence immunoassay (PerkinElmer) and a standard curve previously determined for the PEG-GH conjugate.

[0035] FIG. 1 is an HPLC curve of trypsin map analysis of the reaction of hGH and 40K branched butyrylaldehyde hGH or 40K branched chain aldehyde hGH. The upper panel is a trypsin mapping of 40K branched butyrylaldehyde hGH. The middle panel is a trypsin mapping of the 40K branched chain aldehyde hGH. The lower panel is a trypsin mapping of non-pegylated hGH. T1 is an N-terminal trypsin fragment. [0036] FIG. 2 shows the amino acid sequence of human growth hormone (SEQ ID NO: 1). [0037] FIG. 3 shows the efficacy of 40K branched chain butyraldehyde hGH in a rat weight gain assay. Hypophysectomized female Sprague-Dawley rats were purchased from Harlan Labs at 4-5 weeks of age (100-125 g). In animal cages, animals were weighed daily for 4-10 days to maintain a constant room temperature of 80 ° F. and establish baseline growth in rats. Starting on day 0, control rats (˜100 g) were then given daily subcutaneous injections of ˜0.3 mg / kg hGH (black circles) or PBS (white circles) for 11 consecutive days. The 40K branched-chain butyraldehyde hGH test group (black squares) received a single dose of 1.8 mg / kg of PHA-794428 on days 0 and 6. There were 8-10 animals in each group. Average growth +/- SEM was plotted. [0038] FIG. 4 shows the growth promoting effect in response to a dose of 40K branched chain butyraldehyde hGH in rats. This efficacy study is similar to that described in FIG. 3 except that various single doses of 40K branched chain butyraldehyde hGH were administered (Day 0 only) and the study was followed for 6 days. Went in style. In the control group, once daily injection of either 0.3 mg / kg hGH (black circle) or PBS (white circle) was administered continuously for 6 days. 40K branched butyrylaldehyde hGH is 1.8 mg / kg (black square), 0.6 mg / kg (white square), 0.2 mg / kg (black triangle), or 0.067 mg / kg (white triangle). Administered. 8 animals in each group [0039] FIG. 5 shows tibial growth in response to 40K branched chain butyraldehyde hGH. Hypophysectomized rats were treated as described in FIG. On day 11, the animals were sacrificed, the left tibia was excised, irradiated with X-rays, and the bone length was measured using calipers. Plot the average length +/- SEM. An asterisk indicates a significant difference from the control group (p <0.05). [0040] FIG. 6 shows plasma IGF-1 levels for a 6-day efficacy study. The animals were treated as described in FIG. Blood samples were taken at various times and serum IGF-1 levels were determined by ELISA. The plot is the mean +/− SEM.

Claims (8)

Poly (ethylene glycol) modified hGH having the structure of Formula II:

[Wherein n is an integer between 1 and 10;
m is an integer between 1 and 10;
R is human growth hormone or methionyl growth hormone]
, Alone or in combination with another therapeutic agent, for the prevention and / or treatment of a disease or disorder for which the use of growth hormone is beneficial, wherein the disease or disorder is an erectile function Failure, HIV lipodystrophy, fibromyalgia, osteoporosis, memory impairment, depression, Crohn's disease, skeletal dysplasia, traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, Down syndrome, idiopathic short stature Disease (ISS), end stage renal disease (ESRD), very low birth weight infant (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, short stature due to glucocorticoid treatment in children, and short stature of premature infants Said medicament selected from the group consisting of:   Said diseases or disorders for which the use of growth hormone is beneficial are traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, idiopathic short stature (ISS), very low birth weight infant (VLBW), glucocorticoid treatment in children The medicament according to claim 1, selected from the group consisting of short stature due to and low catch height growth of premature infants.   The medicament according to claim 1 or 2, wherein n is equal to 4 and m is equal to 3.   The medicament according to any one of claims 1 to 3, wherein the human growth hormone comprises the amino acid sequence of SEQ ID NO: 1.   5. The medicament of claim 4, wherein more than 90% of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1.   6. The medicament of claim 5, wherein more than 95% of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1.   The medicament according to any one of claims 1 to 6, wherein the mPEG has a molecular weight of 20 kDa. A human growth hormone-PEG conjugate of formula II in combination with another therapeutic agent and at least one pharmaceutically acceptable carrier:

[Wherein n is an integer between 1 and 10;
m is an integer between 1 and 10;
R is human growth hormone or methionyl growth hormone]
A composition comprising

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